At least 85% of human proteins are considered to be challenging targets for small molecule drugs using conventional discovery approaches, such as high-throughput screening of existing chemical libraries (Hopkins et al., 2002). A particularly important, but historically intractable, subset of these proteins are those that elicit their biological effects through protein-protein interactions (Nero et al., 2014); while some protein-protein interactions consisting of short alpha helical domains inserted into a deep hydrophobic pocket in an interacting protein have been amenable to disruption with small molecules (e.g., the p53-Mdm2 interaction (Vassilev et al., 2004)), many protein-protein interactions have been largely resistant to small molecule inhibition using high-throughput screening of standard chemical libraries. Within this category are the RAS GTPases, which are proposed to be among the most tantalizing and thoroughly validated targets in cancer biology due to their high prevalence and frequent essentiality in lethal malignancies (Downward et al., 2003). RAS gene mutations are found at high rates in three of the top four lethal malignancies in the United States—pancreatic (90%), colon (45%), and lung cancers (35%) (Id.). Many tumors have been shown to be dependent on continued expression of oncogenic RAS proteins in cell and animal models (Weinstein et al., 2008). However, RAS proteins have been viewed as challenging targets, primarily due to the lack of a sufficiently large and deep hydrophobic site for small molecule binding, aside from the GTP-binding site. The picomolar affinity of GTP (John et al., 1990) makes competitive inhibition impractical, in contrast to the ATP-binding site on kinases. For these reasons, traditional high-throughput screening has been unable to provide high affinity small molecule RAS ligands.
The RAS proteins play a central role in a number of signal transduction pathways controlling cell growth and differentiation. They function as a binary switch, transitioning from an inactive GDP-bound state to an active GTP-bound state (Downward et al., 2003). GTP binding enables several residues, primarily in the switch I region (residues 30-40) and the switch II region (residues 60-70) to adopt a conformation that permits RAS effector proteins to bind; this transition is reciprocally regulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). A mutation resulting in the impairment of the intrinsic GTPase activity of RAS proteins, or preventing GAP binding, constitutively activates downstream signaling pathways and contributes to the malignant phenotype. Thus, there exists an unmet need for compounds that selectively bind a RAS protein, particularly an oncogenic mutant of a RAS protein.